Telomerase activating compounds for use in fertility and related applications

ABSTRACT

The present invention is directed to methods and uses of a series of compounds and compositions comprising the same for treating diseases, disorders and/or conditions related to fertility and preserving same and conditions related to same. In some aspects, the compounds and or compositions as herein described promote, improve, recover or restore fertility in a subject in need thereof, including in male and/or female subjects. Such methods/uses include promoting, enhancing or improving fertility-associated cell or tissue yield as part of an in vitro fertilization protocol.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims the benefit of Israel Patent ApplicationNumber 250567, filed Feb. 12, 2017, and U.S. Provisional Application No.62/458,028 filed Feb. 13, 2017 and Israel Patent Application Number257470, filed Feb. 11, 2018, all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention is directed to use of a series of compounds andcompositions comprising the same for treating diseases, disorders and/orconditions related to fertility and preserving same and conditionsrelated to same.

BACKGROUND OF THE INVENTION

Telomerase is a ribonucleoprotein responsible for the elongation ofrepetitive DNA structure in the chromosome ends of eukaryotes—theTelomeres, which shorten every cell division due to the “end replicationproblem”. Telomerase is composed of two major subunits, the catalyticsubunit telomerase reverse transcriptase (TERT) and an RNA component(TR) which serves as a template for telomere elongation. Telomeraseexpression is essential for proliferative cells. One of the majorgenetic mechanisms determining cell proliferative capacity themaintenance of telomeres by the enzyme telomerase.

Telomerase activity has been detected in human germ cells, such as inthe ovary and expressed in intensively dividing cells and tissues whichhave the capacity of regeneration. Decrease in fertility among TERTdeficient animal models and TR deficient female mice have shown uterineatrophy, which indicates dysfunction of granulosa cells. During thedevelopment of oocytes, folliculogenesis is accompanied by significantproliferation of granulosa cells.

Most somatic cells contain low or undedicated expression of telomerasebut during the spermatogenesis process telomerase is active and itscatalytic subunit TERT is expressed, as well.

Beyond the above, little is known about the role of telomerase and itssubunits in developing gonads, for example, in oocytes and granulosacells or in spermatogenesis. Still less is understood in terms of thedifferential impact of telomerase activating compounds and treatment orrecovery of fertility.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides a method of promoting,improving, recovering or restoring fertility in a subject in needthereof, comprising contacting a gonadal or fertility-associated cell ortissue with a compound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof.

In some embodiments, this invention provides for the use of a compoundrepresented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof for promoting,improving, recovering or restoring fertility in a subject in needthereof.

According to these aspects and in some embodiments, such method and/oruse promoting, improving, recovering or restoring fertility in a subjectbeneficially modulates gonadotropin expression or steroid hormoneexpression in terms of timing or quantity or a combination thereof, orin some embodiments, improves follicle maturation in terms of timing orquantity or a combination thereof, or in some embodiments, improvessperm quantity, quality, motility or a combination thereof, or anycombination of same.

In other embodiments, this invention provides a method of promoting,improving, recovering or restoring function to gonadal cells or tissuesin a subject in need thereof, comprising contacting a gonadal orfertility-associated cell or tissue with a compound represented by thestructure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof.

In other embodiments, this invention provides for the use of a compoundrepresented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof for promoting,improving, recovering or restoring function to gonadal cells or tissuesin a subject in need thereof.

In some embodiments, this invention provides for a method of promoting,enhancing or improving fertility-associated cell or tissue yield as partof an in vitro fertilization protocol, comprising contacting saidfertility-associated cell or tissue with a compound represented by thestructure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof.

In still other embodiments, this invention provides for the use of acompound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof for promoting,enhancing or improving fertility-associated cell or tissue yield as partof an in vitro fertilization protocol.

In one embodiment the structure of formula I is represented by thestructure of formula IV:

-   -   wherein R₁, R₃, R₄, R₆, R₇, R₉ and R₁₀ are as described above.

In another embodiment the structure of formula I is represented by thestructure of formula VI:

-   -   wherein R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are the same or        different comprising halogen, aryl, alkyl, cycloalkyl,        heterocycloalkyl, alkoxy, monoalkylamino, dialkylamino or        arylamino; and    -   R₁₀ is as described above.

In another embodiment the structure of formula I is represented by thestructure of formula VII:

In another embodiment the structure of formula I is represented by thestructure of formula VIII:

In another embodiment the structure of formula I is represented by thestructure of formula IX:

In another embodiment the structure of formula I is represented by thestructure of formula X:

In another embodiment the structure of formula I is represented by thestructure of formula XI:

In another embodiment the structure of formula I is represented by thestructure of formula XII:

In another embodiment the structure of formula I is represented by thestructure of formula XIII:

In another embodiment the structure of formula I is represented by thestructure of formula XIV:

In another embodiment the structure of formula I is represented by thestructure of formula XV:

In another embodiment the structure of formula I is represented by thestructure of formula XVI:

In another embodiment, the invention makes use of pharmaceuticalcompositions comprising compounds as described herein for any method asdescribed herein.

In some embodiments, this invention particularly contemplates methods oruses including use of the following compounds:

or any combination of such compounds, and methods/uses employing any ofsuch compounds is to be considered an embodiment of this invention.

In some aspects, the compounds as herein described are suitable foradministration to a female subject, to specifically promote and/orenhance ovulation. In some aspects, such use may, inter alia, includeuse of Compound 79 and Compound 68 and Compound 70. According to thisaspect and in some embodiments, such treatment may increase the numberand/or quality of ovum that ultimately can be fertilized in situ.

In some aspects, the compounds as herein described are suitable foradministration to a female subject, to specifically enhance the qualityand/or number of ovum appropriate for retrieval for an in vitrofertilization or harvest procedure. In some aspects, such use may, interalia, include use of Compound 79 and Compound 68 and Compound 70.

In some aspects, Compound 79 particularly and unexpectedly outperformsother compounds in terms of promoting/enhancing ovulation promotingevents.

In some aspects, the compounds as herein described are suitable foradministration to a female subject, to specifically protect or promoterecovered ovum quality and/or quantity following exposure of suchsubject to irradiation.

In some aspects, the compounds as herein described promote or enhancefertilization events in situ or in vitro.

In some aspects, the compounds as herein described are suitable foradministration to a male subject, to specifically enhance the qualityand/or number of sperm for applications in fertilization in situ or invitro. In some aspects the compounds as herein described may promoteenhanced sperm capacitance, maturity, number, quality, motility or acombination of same.

In some aspects, the compounds as herein described are suitable foradministration to a male subject, to specifically protect or promoterecovered sperm fertilization capacity following exposure of suchsubject to irradiation or as a result of aging or premature aging in asubject.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter regarded as the invention is particularly pointed outand distinctly claimed in the concluding portion of the specification.The invention, however, both as to organization and method of operation,together with objects, features, and advantages thereof, may best beunderstood by reference to the following detailed description when readwith the accompanying drawings in which:

FIG. 1: plots granulosa cells treated with different concentrations ofPMSG for 6 hours. RNA extract served for TERT relative mRNA levelsmeasurement by qRT-PCR with specific primers for TERT and β-actin as areference gene. Results represents average±SE (n=3).

FIG. 2: Activation of telomerase expression by PMSG in rat granulosacells.

FIG. 3: Granulosa cells treated with two embodied compounds, compound 79and compound 70, at 50 nM and 200 nM for 6 hours. Protein extractsserved as the source for telomerase activity measurement using a TRAPassay.

FIG. 4: Granulosa cells treated with two embodied compounds, compound 79and compound 70, at 50 nM and 200 nM for 6 hours. RNA extracts assessedfor TERT relative mRNA levels measurement by qRT-PCR with specificprimers for TERT with β-actin serving as the reference gene. Resultsrepresents average ±SE (n=3). Statistical significance determined byT-TEST *P-value <0.05 **P-value <0.01.

FIG. 5: Granulosa cells treated with two embodied compounds, compound 79and compound 70, at 50 nM for 6 hours. RNA extract evaluated for StArrelative mRNA levels measurement by qRT-PCR using specific primers forStAr and β-actin serving as a reference gene. Results representsaverage±SE (n=3). Statistical significance determined by T-TEST *P-value<0.05.

FIG. 6: Progesterone levels of granulosa cells treated with two embodiedcompounds, compound 79 and compound 70, at 50 nM with or without PMSG 1IU/mL for 24 hours, by evaluation of cell-culture medium byradioimmunoassay (RIA). Value of zero represents un-detectable amount ofprogesterone. Results represents average±SE (n=3). Statisticalsignificance determined by T-TEST *P-value <0.05.

FIG. 7: Granulosa cell viability for samples treated with two embodiedcompounds, compound 79 and compound 70, in 50 nM with 1 IU/mL PMSG for24 hours. Relative cell viability was measured by XTT proliferationassay. Results represents average±SE (n=3).

FIG. 8: Telomerase activity in mouse ovary at different mouse ages, asquantified by TRAP assay of mouse ovary at various ages (10 days, 18days, 1 month and 3 months old) by densitometry analysis using theEZquant software and calculated as % of a constant positive control.(mean±s.e.m.; n=12 mice).

FIG. 9: TERT expression in ovaries derived from 3 months old mice atvarious estrous stages. Total RNA was prepared from the ovaries of 3months old mice at the various estrous stages and qRT-PCR was performedwith the appropriate primers. Data are mean±s.e.m.; n=12.

FIG. 10: Telomerase activity in mouse ovary following treatment with theindicated compounds. Mice were injected s.c with Compound 79 or 68 orDMSO. Twelve hours after treatment, proteins extract were prepared frommice ovary and 1 microg was added to the TRAP specific reaction mixture(A a representative picture). Quantification of telomerase activity (bydensitometric analysis of the TRAP assay DNA products using the EZquantsoftware) in ovary proteins extract derived from mouse at different agesB. 10 days, C. 18 days, D. 1 month E. 3 months. Symbols: PC— positivecontrol (proteins extract of cancer cells). Negative control (NC)contained CHAPS buffer instead of proteins extract. IS=internalstandard. The results are mean±s.e.m. ; n=48 mice) T test: *p<0.05relative to DMSO.

FIG. 11. The compounds of this invention increase telomerase expression(A) and activity (B) in mouse ovary at all estrous stages. Female miceat the various estrous stages were untreated or treated with a singleinjected (s.c ) of: 0.5% DMSO, Compound 79 or 68 at 6 mg/kg. The micewere sacrificed 12 hrs post treatment and the ovaries were removed,A—total RNA was prepared followed by cDNA preparation and analysis byqRT-PCR with the appropriate primers. B— protein extracts were preparedand 1 μg of proteins was analyzed for telomerase activity using the TRAPassay. The telomerase DNA products were quantified by densitometricanalysis with the EZquant software and calculated as % of a constantpositive control. The results are mean±SE, n≥3 mouse per group Ttest,*p<0.05, **p<0.01, ***p<0.001 relative to DMSO.

FIG. 12: (A) Female mice at the various estrous stages were injected s.cwith DMSO, Compound 79 or 68. After 12 hours the mice were sacrificedand the ovaries were removed and weighted. The results are means±SE, n≥6ovaries per group. (B) A Representative picture of ovaries from 3 monthsold mice 12 hrs after treatments.

FIG. 13: Vaginal smears at proestrus and estrus stages before and after12 h of treatment with the indicated compounds. Vaginal smears weretaken from female mice following by s.c. injection with DMSO, Compound79 or 68.(A). Before treatment proestrus stages (A), estrus stages (C).After 12 hours of treatment the vaginal smears were taken again.proestrus stages (B), estrus stages (D). The vaginal smears were stainedby crystal violet.

FIG. 14: Hematoxylin-eosin staining of mice ovary at the various estrousstages. Mice at the various estrous stages were injected s.c with DMSO,or with one of compounds 68, 70 and 79, as indicated. After 12 hours themice were sacrificed and ovaries were removed and fixed withformaldehyde. The tissues were stained with Hematoxylin-eosin andvisualized by light microscopy ×4. CL—corpus luteum, GC—granulosa cells,n≥3 per group.

FIG. 15: PCNA staining of mouse ovary following treatments with embodiedcompounds: representative pictures. Mice at the estrus stage (A) or atthe metestrus stage (B) were injected s.c with DMSO, or with one ofCompound 79 or 68. After 12 hours the mice were sacrificed and theovaries were removed and fixed with formaldehyde. The tissues weresubjected to immunofluorescence analysis using anti-PCNA antibody.Symbols: CL—corpus luteum, GC-granulosa cells.

FIG. 16: Progesterone concentrations in the plasma of female mouse 12hours post treatment with the embodied compounds. Mice at the variousestrous stages were treated with the indicated compounds or vehicle.Mice were sacrificed 12 hrs post treatment and Blood samples were takenand the concentration of progesterone was measured as described(mean±s.e.m. ; n=34 mice). (A) P+12 h, (B) E+12 h, (C) M+12 h, (D) D+12h,

FIG. 17: The compounds in accordance with this invention increase thenumber of embryos. ICR mice were treated with a single s.c. injection ofthe indicated compounds, 6 mg/kg or with DMSO 0.5%. Immediately afterthe injection female mouse was put into a cage of the male mouse, after14 days female mouse was sacrificed and number of embryos counted. Theresults are mean±SE, n≥3 mouse per group. t-test, *p<0.05,**p<0.01,relative to DMSO.

FIG. 18: Telomerase activity in Granulosa cells obtained from Womanundergo IVF procedure. The telomerase DNA products obtained by the TRAPassay FIG. 18A—for 0.5 μg protein of Whole cell (WC) extract and FIG.18B—0.1 μg protein of DNA bound (DB) extract, were analyzed bydensitometry using the EZquant software. Telomerase activity wascalculated as % of the data obtained for a permanent positive control(protein extracts from glioblastoma cells). The results are means±SD ofat least 3 independent TRAP assays.

FIG. 19 Telomerase activity as correlated with Estrogen levels. FIG.19(A) (WC: p=0.01, DB: p=0.42) n=9 for high, n=12 for low, averageenzyme activities of telo (in WC and DB extracts) were calculated forhigh and low estrogen levels. High estrogen level was determined as<1500 ng/ml.

FIG. 20: Correlation between diagnosis of infertility andTelo/activities, n=9 for female infertility, n=12 for male infertility.FIG. 20A—Telo average activity (p=0.159).

FIG. 21: WC protein extracts were analyzed by ELISA kit (EIAab). FIG.21A—Telomerase expression was calculated as % of the total proteinexpression with correlation to blood estrogen levels. FIG.21B—Telomerase expression of individual women.

FIG. 22: H&E staining shows a significant destruction in the morphologyof the testicular tissue particularly in the spermatogonia cells layer.Compound 68 treatment protects the spermatogonia cells layer from thedamaging effects of X-ray and the morphology of the testicular tissueremains intact. ICR mice (3 months old) were subjected to X-rayradiation (2.5 Gy) followed by immediately injection of DMSO ortreatment with Compound 68. The testes were removed 12 hrs aftertreatments and subjected to histological examination, a representativepicture (stained with H&E).

FIG. 23: Examination of CREM expression in the testis before and afterX-ray irradiation, revealed a significant alteration in the expressionpattern in X-ray treated testis compared to the untreated mice. Compound68 and Compound 70 treatments restored the expression of thespermatogenesis markers. ICR mice (3 months old) were subjected to X rayradiation (2.5 Gy) followed by immediately injection of DMSO or Compound68 (a) and Compound 70 (b).The testes were removed 12 hrs aftertreatments and stained with CREM marker.

FIG. 24: IF analysis of γ-H2AX (green) marker showed a significantincrease of DSB formation 12 hrs post-irradiation, compared to UTsamples. In addition, treatment with Compound 70 only did not cause DNAdamages. In irradiated mice, Compound 70 treatment demonstrated adecreased in the expression levels of γ-H2AX in the various cell types,mainly in spermatogonia, indicating that Compound 70 treatment protectsthe testis tissue from DNA damages induced by X-ray radiation. ICR mice(3 months old) were subjected to X-ray radiation (2.5 Gy) followed byimmediate injection of Compound 70. For control, mice were treated withthe compound without irradiation. The testes were removed 12 hrs aftertreatments and stained with γ-H2AX antibody.

FIGS. 25A, 25B, 25C and 25D: Measurement of sperm cells number from theepididymis in non-radiated mice followed by the indicated treatmentsshows a significant increase 12 hrs. post-Compound 68 and Compound 70treatments compared to DMSO, respectively. Sperm count from X-raytreated and untreated mice revealed a significant reduction in spermcount, while treatment of X-ray irradiated mice with the indicatedcompounds significantly increased the numbers of sperms in theepididymis by 1.5-2.7 folds 12 hrs post-radiation (FIGS. 25A and 25B).Treatment of irradiated mice with Compound 68 increased sperm count at 9and 30 days post irradiation (FIGS. 25C and 25D).

FIGS. 26A and 26B: Treatment with embodied compounds protected spermagainst defective head morphology post-radiation exposure. Three-monthold mice were irradiated with X-ray (2.5 Gy) and treated with a singleS.C. injection of 6 mg/kg of compound 68 or 70. Mice were sacrificed 12hours later and sperm were removed from the epididymis and stained witheosin. The number of morphologically defective sperm (head morphology)were quantified and presented as a percentage value of the total spermcount (N=6 mice per group). FIG. 26B provides a light micrograph showinghealthy and defective sperm head morphology observed.

FIGS. 27A and 27B: Embodied compound 70 treatment increased sperm countsin old mice (16 months old) even after a single injection (6 mg/Kg). n=6mice per group, t test.

FIGS. 28A, 28B and 28C: Ovarian tissue histopathology followingirradiation, followed by treatment with Compound 70 or DMSO carrier.

FIGS. 29A, 29B, 29C and 29D: The embodied compounds affect theexpression of gonadotropin hormones in pituitary gland cells. Cells(LβT2) derived from the mouse pituitary gland were assessed for theirsecretion of LH and FSH at 6 (FIGS. 29A and 29B) and 12 hours (FIGS. 29Cand 29D) following treatment with the indicated embodied compounds (50nM). FSH and LH expression was determined by RT-PCR, and quantified from5 different experiments, as assessed for significance, using the t test,one way ANOVA.

FIGS. 30A, 30B, 30C, 30D, 30E and 30F. The embodied compounds affect theexpression of gonadotropin hormones in vivo in female reproductivetissue. Uterine tissue expression of telomerase correlates withgonadotropin expression and the embodied compounds accelerateresponsiveness/progression through the cycle. Assessment of telomeraseversus LH and FSH expression in different states Pro-estrus (P), estrus(E) and met-estrus (M) in compound 534 treated and control female miceis shown.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the invention.However, it will be understood by those skilled in the art that thepresent invention may be practiced without these specific details. Inother instances, well-known methods, procedures, and components have notbeen described in detail so as not to obscure the present invention.

The present invention relates, in some embodiments, to the use of anovel class of tri-phenyl compounds and compositions comprising the samefor the treatment of, inter alia, fertility-related orinfertility-related diseases or conditions capable of being affected byenhanced telomerase expression and/or telomerase activation.

In some aspects, the invention makes use of such compounds whichstimulate and/or increase telomerase expression and/or activity in thegonadal or fertility related cells and tissues of a subject, where theactivity is decreased, missing, altered or normal. Such disordersinclude, inter alia, a) fertility-related impairment, includingimpairment of tissue turnover, which occur with cancer or cancertherapy, b) luteal phase defect; c) premature ovarian failure (primaryovarian insufficiency or hypergonadotropic hypogonadism); and/or d)increasing telomerase expression and/or activity in gonadal orfertility-associated healthy tissue, thus promoting or restoringfertility in the subject. In some aspects, the subject has been exposedto radiation.

In some aspects, the compounds as herein described are suitable foradministration to a female subject, to specifically promote and/orenhance ovulation. In some aspects, such use may include use of anycompound as herein described. According to this aspect and in someembodiments, such treatment may increase the number and/or quality ofovum that ultimately can be fertilized in situ.

In some aspects, the compounds as herein described are suitable foradministration to a female subject, to specifically enhance the qualityand/or number of ovum appropriate for retrieval for an in vitrofertilization or harvest procedure. In some aspects, such use may, interalia, include use of any compound as herein described.

In some aspects, certain compounds as herein described particularly andunexpectedly outperform other compounds in terms of promoting/enhancingovulation promoting events.

In some aspects, the compounds as herein described are suitable foradministration to a female subject, to specifically protect or promoterecovered ovum quality and/or quantity following exposure of suchsubject to irradiation.

In some aspects, the compounds as herein described promote or enhancefertilization events in situ or in vitro.

In some aspects, the compounds as herein described are suitable foradministration to a male subject, to specifically enhance the qualityand/or number of sperm for applications in fertilization in situ or invitro. In some aspects the compounds as herein described may promoteenhanced sperm capacitance, maturity, number, quality, motility or acombination of same.

Compounds of the Invention

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula II:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁, R₃, R₄, R₆, R₇ and R₉ are the same or different, H, D, OH, halogen,nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substitutedketone, —COOH, ester, trifluoromethyl, amide, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl,arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl,alkylhaloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino,alkylamido, arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment Z is carbon. In another embodiment R₁₀ is a methylgroup. In another embodiment R₁, R₃, R₄, R₆, R₇, and R₉ are—(CH₂)_(n)-heterocycloalkyl group, wherein n is between 1-6. In anotherembodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)-aminoalkyl group,wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, andR₉ are —(CH₂)_(n)-dialkylamino group, wherein n is between 1-6 Inanother embodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)—N(CH₃)₂group, wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆,R₇, and R₉ are —(CH₂)_(n)—N(Et)₂ group, wherein n is between 1-6. Inanother embodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)-aryl group,wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, andR₉ are —(CH₂)_(n)-heteroaryl group, wherein n is between 1-6. In anotherembodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)-haloalkyl group,wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, andR₉ are —(CH₂)_(n)-alkoxy group, wherein n is between 1-6. In anotherembodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)-ethoxy group,wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, andR₉ are —(CH₂)_(n)-cycloalkyl group, wherein n is between 1-6.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula III:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R′, R″ and R′″ are independently the same or different comprisinghydrogen, alkyl, haloalkyl, alkylamino, phenyl, benzyl, alkanyloyl,acetyl or benzoyl;

R₁, R₃, R₄, R₆, R₇ and R₉ are the same or different, H, D, OH, halogen,nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substitutedketone, —COOH, ester, trifluoromethyl, amide, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl,arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl,alkylhaloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino,alkylamido, arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula IV:

wherein R₁, R₃, R₄, R₆, R₇, R₉ and R₁₀ are as defined above; or itsisomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula V:

wherein

R′, R″, R′″ are independently the same or different comprising hydrogen,alkyl, haloalkyl, phenyl, benzyl, alkanyloyl, acetyl or benzoyl;

R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are the same or different comprisinghalogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino,monoalkylamino, dialkylamino or arylamino group; and R₇ is as describedabove; or its isomer, pharmaceutically acceptable salt, pharmaceuticalproduct, hydrate, N-oxide, crystal or any combination thereof, andcompositions comprising the same.

In one embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are dialkylaminogroup. In another embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ aredimethylamino group. In another embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, andR₉′ are diethylamino group. In another embodiment, R₁′, R₃′, R₄′, R₆′R₇′, and R₉′ are N-piperidine group. In another embodiment, R₁′, R₃′,R₄′, R₆′ R₇′, and R₉′ are N-pyrolidine group. In another embodiment,R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are N-piperazine group. In anotherembodiment, R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are N-piperazine-4-methylgroup. In another embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ areN-morpholine group. In another embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, andR₉′ are ethoxy group.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula VI:

wherein R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are the same or differentcomprising halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy,amino, monoalkylamino, dialkylamino or arylamino group; and R₁₀ is asdescribed above; or its isomer, pharmaceutically acceptable salt,pharmaceutical product, hydrate, N-oxide, crystal or any combinationthereof, and compositions comprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula VII:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula VIII:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula IX:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula X:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula XI:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula XII:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula XIII:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In another embodiment the structure of formula I is represented by thestructure of formula XIV:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In another embodiment the structure of formula I is represented by thestructure of formula XV:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In another embodiment the structure of formula I is represented by thestructure of formula XVI:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

The term “alkyl” refers, in one embodiment, to a saturated aliphatichydrocarbon, including straight-chain, branched-chain and cyclic alkylgroups. In one embodiment, the alkyl group has 1-12 carbons. In anotherembodiment, the alkyl group has 1-7 carbons. In another embodiment, thealkyl group has 1-6 carbons. In another embodiment, the alkyl group has1-7 carbons. In another embodiment, the alkyl group has 2-6 carbons. Inanother embodiment, the alkyl group has 1-7 carbons. In anotherembodiment, the alkyl group has 2-8 carbons. In another embodiment, thealkyl group has 3-6 carbons. In another embodiment, the alkyl group has3-7 carbons. In another embodiment, the alkyl group has 1-4 carbons. Inanother embodiment, the branched alkyl is an alkyl substituted by alkylside chains of 1 to 5 carbons. In another embodiment, the branched alkylis an alkyl substituted by haloalkyl side chains of 1 to 5 carbons. Thealkyl group may be unsubstituted or substituted by a halogen, haloalkyl,hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro,cyano, amino, monoalkylamino, dialkylamino, carboxyl, thio and/orthioalkyl.

An “alkenyl” group refers, in one embodiment, to an unsaturatedhydrocarbon, including straight chain, branched chain and cyclic groupshaving one or more double bonds. The alkenyl group may have one doublebond, two double bonds, three double bonds, etc. In another embodiment,the alkenyl group has 2-12 carbons. In another embodiment, the alkenylgroup has 2-6 carbons. In another embodiment, the alkenyl group has 2-4carbons. In another embodiment the alkenyl group is ethenyl (CH═CH₂).Examples of alkenyl groups are ethenyl, propenyl, butenyl, cyclohexenyl,etc. The alkenyl group may be unsubstituted or substituted by a halogen,hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro,cyano, amino, monoalkylamino, dialkylamino, carboxyl, thio and/orthioalkyl.

An alkynyl” group refers, in one embodiment, to an unsaturatedhydrocarbon, including straight chain, branched chain and cyclic groupshaving one or more triple bonds. The alkynyl group may have one triplebond, two triple bonds, triple double bonds, etc. In another embodiment,the alkynyl group has 2-12 carbons. In another embodiment, the alkynylgroup has 2-6 carbons. In another embodiment, the alkenyl group has 2-4carbons. In another embodiment the alkynyl group is ethynyl (—CH—CH₂).Examples of alkynyl groups are ethynyl, propynyl, butynyl, cyclohexynyl,etc. The alkynyl group may be unsubstituted or substituted by a halogen,hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro,cyano, amino, monoalkylamino, dialkylamino, carboxyl, thio and/orthioalkyl.

An “alkoxy” group refers, in another embodiment to an alkyl group asdefined above, which is linked to oxygen. Examples of alkoxy groups areethoxy, propoxy, tert-butoxy etc.

A “haloalkyl” group refers, in one embodiment, to an alkyl group asdefined above, which is substituted by one or more halogen atoms, e.g.by F, Cl, Br or I.

An “aryl” group refers, in another embodiment, to an aromatic grouphaving at least one carbocyclic aromatic group or heterocyclic aromaticgroup, which may be unsubstituted or substituted by one or more groupsselected from halogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido,alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino,dialkylamino, carboxy or thio or thioalkyl. In another embodiment, thearyl group is between 4-12-membered ring(s). In another embodiment, thearyl group is between 6-18-membered ring(s). In another embodiment, thearyl group is between 4-8-membered ring(s). In another embodiment, thearyl group is a 6-membered ring. In another embodiment, the aryl groupis a fused ring system comprising of between 2-3 rings. Nonlimitingexamples of aryl rings are phenyl, naphthyl, pyranyl, pyrrolyl,pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl,thiazolyl, imidazolyl, isoxazolyl, and the like.

A “heteroaryl” group refers, in another embodiment, to an aromatic grouphaving at least one heterocyclic aromatic group, which may beunsubstituted or substituted by one or more groups selected fromhalogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido,dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxyor thio or thioalkyl. In another embodiment, the heteroaryl group isbetween 4-12-membered ring(s). In another embodiment, the heteroarylgroup is between 6-18-membered ring(s). In another embodiment, theheteroaryl group is between 4-8-membered ring(s). In another embodiment,the heteroaryl group is a 6-membered ring. In another embodiment, theheteroaryl group is a fused ring system comprising of between 2-3 rings.Nonlimiting examples of heteroaryl rings are pyrrolyl, thienyl,thiazolyl, benzothienyl, naphthothienyl, purinyl, isothiazolyl, furyl,furazanyl, isobenznzofuranyl, pyranyl, chromenyl, xanthenyl,phenoxyxanthiinyl, indolyl, isoindolyl, indolizinyl, isoindolyzinyl,benzothienyl, oxazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, and the like.

A “hydroxyl” group refers, in one embodiment, to an OH group. In someembodiments, when R₁, R₂ or R₃ of the compounds of the present inventionis OR, then R is not OH.

In one embodiment, the term “halo” refers to a halogen, such as F, Cl,Br or I.

In another embodiment, the phrase “phenol” refers to an alcohol (OH)derivative of benzene.

An “amino” group refers to, in one embodiment, to a nitrogen atomattached by single bonds to hydrogen atoms, alkyl groups, alkenyl groupsor aryl groups as described above, as described above, or a combinationthereof. Nonlimiting examples of amino groups are NH₂, N(Me)₂, N(Et)₂,N(Ph)₂ and the like.

A “cycloalkyl” group refers, in one embodiment, to a non-aromatic,monocyclic or polycyclic ring comprising carbon and hydrogen atoms. Acycloalkyl group can have one or more carbon-carbon double bonds in thering so long as the ring is not rendered aromatic by their presence.Examples of cycloalkyl groups include, but are not limited to,(C₃-C₇)cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenesand (C₃-C₇)cycloalkenyl groups, such as cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclohexenyl, and cycloheptenyl, and unsaturated cyclicand bicyclic terpenes. Preferably, the cycloalkyl group is a monocyclicring or bicyclic to a ring structure comprising in addition to carbonatoms, sulfur, oxygen, nitrogen or any combination thereof, as part ofthe ring. In another embodiment the cycloalkyl is a 3-12-membered ring.In another embodiment the cycloalkyl is a 6-membered ring. In anotherembodiment the cycloalkyl is a 5-7-membered ring. In another embodimentthe cycloalkyl is a 4-8-membered ring. In another embodiment, thecycloalkyl group may be unsubstituted or substituted by a halogen,haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido,cyano, nitro, CO₂H, amino, monoalkylamino, dialkylamino, carboxyl, thioand/or thioalkyl.

A “heterocycloalkyl” group refers, in one embodiment, to a non-aromatic,monocyclic or polycyclic ring comprising carbon and in addition tocarbon, sulfur, phosphor, oxygen or nitrogen, as part of the ring. Aheterocycloalkyl group can have one or more double bonds in the ring solong as the ring is not rendered aromatic by their presence. Examples ofheterocycloalkyl groups include, but are not limited to, piperidine,piperazine, pyrane, morpholine. Preferably, the heterocycloalkyl groupis a monocyclic ring or bicyclic to a ring structure comprising inaddition to carbon atoms, sulfur, oxygen, nitrogen or any combinationthereof, as part of the ring. In another embodiment the heterocycloalkylis a 3-12-membered ring. In another embodiment the heterocycloalkyl is a6-membered ring. In another embodiment the heterocycloalkyl is a5-7-membered ring. In another embodiment the heterocycloalkyl is a4-8-membered ring. In another embodiment, the heterocycloalkyl group maybe unsubstituted or substituted by a halogen, haloalkyl, hydroxyl,alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO₂H,amino, monoalkylamino, dialkylamino, carboxyl, thio and/or thioalkyl. Inanother embodiment the heterocycloalkyl is a cyclic urea, imidazolinyl,imidazolidinyl, pyrrolinyl, pyrrolidinyl, oxazolinyl, isoxazolinyl,oxazolidinyl, oxazolidonyl, isoxazolidonyl, pyrazolinyl, pyrazolidinyl,piperidyl, piperazine, morpholinyl.

The terms “alkylalkoxy”, “alkylhaloalkyl”, “alkylaryl”,“alkylcycloalkyl”, “alkylheterocycloalkyl”, “alkylheteroaryl” and“alkylamino” refer, in one embodiment, to an alkyl group, as definedabove, linked to alkoxy, haloalkyl, aryl, cycloalkyl, heterocycloalkyl,heteroaryl or amino group, respectively. The alkoxy, haloalkyl, aryl,cycloalkyl, heterocycloalkyl, heteroaryl or amino groups are as definedhereinabove. Examples include, but are not limited to, CH₂—OEt,CH₂—N-piperidine, CH₂—N-piperazine, CH₂—N(Me)₂, etc.

In another embodiment, the fused heterocycloalkyl of formula I-IV withthe main aromatic ring forms a phenylpyrrolidone group. In anotherembodiment, the fused aryl of formula I-IV, with the main aromatic ringforms a naphthalene group. In another embodiment, the fused heteroarylof formula I-IV, with the main aromatic ring forms a quinoline orisoquinoline group.

In one embodiment, this invention provides for the use of a compound asherein described and/or, its analog, derivative, isomer, metabolite,pharmaceutically acceptable salt, pharmaceutical product, hydrate,N-oxide, prodrug, polymorph, impurity or crystal or combinationsthereof.

In one embodiment, the term “isomer” includes, but is not limited to,optical isomers and analogs, structural isomers and analogs,conformational isomers and analogs, and the like.

In one embodiment, the term “isomer” is meant to encompass opticalisomers of the tri-phenyl compound. It is to be understood that thepresent invention encompasses any racemic, optically-active,polymorphic, or stereoisomeric form, or mixtures thereof, which formpossesses properties useful in the treatment of telomerase expressionand/or activity conditions described herein. In one embodiment, thetri-phenyl compounds are the pure (R)-isomers. In another embodiment,the tri-phenyl compounds are the pure (S)-isomers. In anotherembodiment, the tri-phenyl compounds are a mixture of the (R) and the(S) isomers. In another embodiment, the tri-phenyl compounds are aracemic mixture comprising an equal amount of the (R) and the (S)isomers. It is well known in the art how to prepare optically-activeforms (for example, by resolution of the racemic form byrecrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase).

The invention includes “pharmaceutically acceptable salts” of thecompounds of this invention, which may be produced, in one embodiment,to form alkali metal salts and to form addition salts of free acids orfree bases. Suitable pharmaceutically-acceptable acid addition salts ofcompounds of this invention may be prepared from an inorganic acid orfrom an organic acid. In one embodiment, examples of inorganic acids arehydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric andphosphoric acid. In one embodiment, organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, oxalic, p-toluenesulphonic,mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethylsulfonic, benzenesulfonic, sulfanilic,stearic, cyclohexylaminosulfonic, algenic, galacturonic acid. In oneembodiment, suitable pharmaceutically-acceptable base addition salts ofcompounds of this invention include metallic salts made from aluminum,calcium, lithium, magnesium, potassium, sodium and zinc or organic saltsmade from N,N′-dibenzylethyleneldiamine, choline, chloroprocaine,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocain. All of these salts may be prepared by conventional means fromthe corresponding compounds.

Pharmaceutically acceptable salts can be prepared, from the phenoliccompounds, in other embodiments, by treatment with inorganic bases, forexample, sodium hydroxide. In another embodiment, esters of the phenoliccompounds can be made with aliphatic and aromatic carboxylic acids, forexample, acetic acid and benzoic acid esters.

The invention also includes use of N-oxides of the amino substituents ofthe compounds described herein.

This invention provides for the use of derivatives of the compounds asherein described. In one embodiment, “derivatives” includes but is notlimited to ether derivatives, acid derivatives, amide derivatives, esterderivatives and the like. In another embodiment, this invention furtherincludes use of hydrates of the compounds as described herein. In oneembodiment, “hydrate” includes but is not limited to hemihydrate,monohydrate, dihydrate, trihydrate and the like.

This invention provides, in other embodiments, use of metabolites of thecompounds as herein described. In one embodiment, “metabolite” means anysubstance produced from another substance by metabolism or a metabolicprocess.

This invention provides, in other embodiments, use of pharmaceuticalproducts of the compounds as herein described. The term “pharmaceuticalproduct” refers, in other embodiments, to a composition suitable forpharmaceutical use (pharmaceutical composition), for example, asdescribed herein.

In some embodiments, the invention provides methods and uses of thecompounds and/or compositions comprising the compound of this invention,for promoting, improving, recovering or restoring fertility in a subjectin need thereof, comprising contacting a gonadal or fertility-associatedcell or tissue with a compound. In some embodiments, the inventionprovides a method of promoting, improving, recovering or restoringfunction to gonadal cells or tissues in a subject in need thereof,comprising contacting a gonadal or fertility-associated cell or tissuewith a compound as herein descried and/or a composition comprising same.the invention provides methods and uses of the compounds and/orcompositions comprising the compound of this invention, for promoting,enhancing or improving fertility-associated cell or tissue yield as partof an in vitro fertilization protocol.

In some embodiments, the invention provides compositions comprising thecompound of this invention and/or treating female infertility-relatedconditions including luteal phase defect or premature ovarian failure(primary ovarian insufficiency or hypergonadotropic hypogonadism);and/or diminished granulosa cell telomerase activity; and/or treatingmale infertility-related conditions including impaired sperm productionor impaired sperm delivery; and/or as an adjunct to in vitrofertilization (IVF) techniques; and/or to enhance sperm quality and/oregg quality. For example, and in some embodiments, the compounds of thisinvention prolong blastocyst viability in ex vivo culture, which in turnenhances implantation efficiency. In some embodiments, the treatment ofthe population with the compounds as herein described renders them morereceptive to other IVF therapeutics, or in some embodiments, allows forthe evaluation of combination therapies, or new compounds.

In some embodiments, the invention provides any compound as hereindescribed or combinations of same or any composition comprising thecompound or combinations of compounds of this invention in restoring,enhancing, rescuing or promoting any aspect of fertility in a subjectexposed to radiation.

In one embodiment, this invention provides methods of treatment using acompound of this invention, or composition comprising the same, asherein described. In some embodiments, the invention provides methods ofuse of a compound of this invention for the treatment of the indicateddiseases, disorders or conditions, and includes use of compositionscomprising the same.

It will be appreciated that it is contemplated herein to use anycombination of any compounds as herein described and/or compositionscontaining such combinations of compounds as herein described, for usein any method or assay, etc. as herein described.

In one embodiment, the terms “treating” or “treatment” includespreventive as well as disorder remittive treatment. The terms“reducing”, “suppressing” and “inhibiting” have their commonlyunderstood meaning of lessening or decreasing, in another embodiment, ordelaying, in another embodiment, or reducing, in another embodiment theincidence, severity or pathogenesis of a disease, disorder or condition.In embodiment, the term treatment refers to delayed progression of,prolonged remission of, reduced incidence of, or amelioration ofsymptoms associated with the disease, disorder or condition. In oneembodiment, the terms “treating” “reducing”, “suppressing” or“inhibiting” refer to a reduction in morbidity, mortality, or acombination thereof, in association with the indicated disease, disorderor condition. In one embodiment, the term “progression” refers to anincreasing in scope or severity, advancing, growing or becoming worse.The term “recurrence” means, in another embodiment, the return of adisease after a remission. In one embodiment, the methods of treatmentof the invention reduce the severity of the disease, or in anotherembodiment, symptoms associated with the disease, or in anotherembodiment, reduces the number of biomarkers expressed during disease.

In one embodiment, the term “treating” and its included aspects, refersto the administration to a subject with the indicated disease, disorderor condition, or in some embodiments, to a subject predisposed to theindicated disease, disorder or condition. The term “predisposed to” isto be considered to refer, inter alia, to a genetic profile or familialrelationship which is associated with a trend or statistical increase inincidence, severity, etc. of the indicated disease. In some embodiments,the term “predisposed to” is to be considered to refer, inter alia, to alifestyle which is associated with increased risk of the indicateddisease. In some embodiments, the term “predisposed to” is to beconsidered to refer, inter alia, to the presence of biomarkers which areassociated with the indicated disease, for example, in cancer, the term“predisposed to” the cancer may comprise the presence of precancerousprecursors for the indicated cancer.

The term “administering”, in another embodiment, refers to bringing asubject in contact with a compound of the present invention.Administration can be accomplished in vitro, i.e. in a test tube, or invivo, i.e. in cells or tissues of living organisms, for example humans.In one embodiment, the present invention encompasses administering thecompounds of the present invention to a subject.

In one embodiment, the methods of this invention make use of thedescribed compound of this invention contacting or binding a telomeraseenzyme in an amount effective to increase telomerase activity and/orexpression and thereby mediating the described effects. In someembodiments, the methods of this invention may include the preliminarystep of identifying a cell or tissue in which an increase telomeraseactivity and/or expression is desired. The cell may be in culture, i.e.in vitro or ex vivo, or within a subject or patient in vivo. In oneembodiment, an increase in telomerase expression and/or activity in acell or tissue includes, for example, enhancement of the replicativecapacity and/or lifespan of the contacted cells.

In some embodiments, for any method/kit or application as hereindescribed, the invention specifically contemplates use of compounds 68,70 and 79, respectively:

respectively

In some aspects, such cells may specifically be protected, enhanced,stimulated, etc. following exposure to radiation.

Pharmaceutical Compositions

In some embodiments, this invention provides methods of use whichcomprise administering a composition comprising the described compounds.As used herein, “pharmaceutical composition” means a “therapeuticallyeffective amount” of the active ingredient, i.e. the compounds of thisinvention, together with a pharmaceutically acceptable carrier ordiluent. A “therapeutically effective amount” as used herein refers tothat amount which provides a therapeutic effect for a given conditionand administration regimen.

In some embodiments, this invention provides compositions which maycomprise at least one compound of this invention, in any form orembodiment as described herein. In some embodiments, the term “a” is tobe understood to encompass a single or multiple of the indicatedmaterial. In some embodiments, the term “a” or “an” refers to at leastone.

In some embodiments, any of the compositions of this invention willconsist of a compound of this invention, in any form or embodiment asdescribed herein. In some embodiments, of the compositions of thisinvention will consist essentially of a compound of this invention, inany form or embodiment as described herein.

In some embodiments, the term “comprise” refers to the inclusion of theindicated active agent, such as the compounds of this invention, as wellas inclusion of other active agents, and pharmaceutically acceptablecarriers, excipients, emollients, stabilizers, etc., as are known in thepharmaceutical industry. In some embodiments, any of the compositions ofthis invention will comprise a compound of formula I-XVI in any form orembodiment as described herein. In some embodiments, any of thecompositions of this invention will consist of a compound of formulaI-XVI, in any form or embodiment as described herein. In someembodiments, of the compositions of this invention will consistessentially of a compound of this invention, in any form or embodimentas described herein. In some embodiments, the term “comprise” refers tothe inclusion of the indicated active agent, such as the compound ofthis invention, as well as inclusion of other active agents, andpharmaceutically acceptable carriers, excipients, emollients,stabilizers, etc., as are known in the pharmaceutical industry. In someembodiments, the term “consisting essentially of” refers to acomposition, whose only active ingredient is the indicated activeingredient, however, other compounds may be included which are forstabilizing, preserving, etc. the formulation, but are not involveddirectly in the therapeutic effect of the indicated active ingredient.In some embodiments, the term “consisting essentially of” refers to acomposition, whose only active ingredient with a comparable mode ofaction, or comparable molecular target is the indicated activeingredient, however, other active ingredients may be incorporated, withsuch secondary active ingredients acting on different targets, or in apalliative capacity. In some embodiments, the term “consistingessentially of” may refer to components which facilitate the release ofthe active ingredient. In some embodiments, the term “consisting” refersto a composition, which contains a compound as herein described as theonly active ingredient and a pharmaceutically acceptable carrier orexcipient.

In another embodiment, the invention provides a composition comprising acompound of this invention, as herein described, or its prodrug, analog,isomer, metabolite, derivative, pharmaceutically acceptable salt,pharmaceutical product, polymorph, crystal, impurity, N-oxide, ester,hydrate or any combination thereof and a suitable carrier or diluent.

An active component can be formulated into the composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts, which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed from the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The pharmaceutical compositions containing the compound of thisinvention can be administered to a subject by any method known to aperson skilled in the art, such as orally, parenterally,intravascularly, paracancerally, transmucosally, transdermally,intramuscularly, intranasally, intravenously, intradermally,subcutaneously, sublingually, intraperitoneally, intraventricularly,intracranially, intravaginally, by inhalation, rectally, intratumorally,or by any means in which the recombinant virus/composition can bedelivered to tissue (e.g., needle or catheter). Alternatively, topicaladministration may be desired for application to mucosal cells, for skinor ocular application. Another method of administration is viaaspiration or aerosol formulation.

The compositions of the present invention are formulated in oneembodiment for oral delivery, wherein the active compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. The tablets, troches, pills, capsules and the like mayalso contain the following: a binder, as gum tragacanth, acacia,cornstarch, or gelatin; excipients, such as dicalcium phosphate; adisintegrating agent, such as corn starch, potato starch, alginic acidand the like; a lubricant, such as magnesium stearate; and a sweeteningagent, such as sucrose, lactose or saccharin may be added or a flavoringagent, such as peppermint, oil of wintergreen, or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. Syrup of elixir may contain the activecompound, sucrose as a sweetening agent methyl, and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Inaddition, the active compounds may be incorporated intosustained-release, pulsed release, controlled release or postponedrelease preparations and formulations.

In another embodiment, the compositions of this invention comprise oneor more, pharmaceutically acceptable carrier materials.

In one embodiment, the carriers for use within such compositions arebiocompatible, and in another embodiment, biodegradable. In otherembodiments, the formulation may provide a relatively constant level ofrelease of one active component. In other embodiments, however, a morerapid rate of release immediately upon administration may be desired. Inother embodiments, release of active compounds may be event-triggered.The events triggering the release of the active compounds may be thesame in one embodiment, or different in another embodiment. Eventstriggering the release of the active components may be exposure tomoisture in one embodiment, lower pH in another embodiment, ortemperature threshold in another embodiment. The formulation of suchcompositions is well within the level of ordinary skill in the art usingknown techniques. Illustrative carriers useful in this regard includemicroparticles of poly (lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other illustrativepostponed-release carriers include supramolecular biovectors, whichcomprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as phospholipids. The amount ofactive compound contained in one embodiment, within a sustained releaseformulation depends upon the site of administration, the rate andexpected duration of release and the nature of the condition to betreated suppressed or inhibited.

In one embodiment it will be desirable to deliver the compositionsdisclosed herein parenterally, intravenously, intramuscularly, or evenintraperitoneally. Such approaches are well known to the skilledartisan, some of which are further described, for example, in U.S. Pat.Nos. 5,543,158; 5,641,515 and 5,399,363, all of which are fullyincorporated by reference. In certain embodiments, solutions of theactive compounds as free base or pharmacologically acceptable salts maybe prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions may also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi.

In another embodiment, it will be preferable to include isotonic agents,for example, sugars or sodium chloride. In other embodiments, prolongedabsorption of the injectable compositions will be desirable. Prolongedabsorption of the injectable compositions can be brought about by theuse of agents delaying absorption, for example, aluminum monostearateand gelatin, in the compositions.

Parenteral vehicles include in certain embodiments sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's and fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, antioxidants, collatingagents, inert gases and the like

In some embodiments, the compounds of this invention may be administeredat various dosages to a subject, which in one embodiment, is a humansubject. In one embodiment, the compounds of this invention areadministered at a dosage of 0.1-200 mg per day. In one embodiment, thecompound of this invention is administered at a dose of 0.1-10 mg, or inanother embodiment, 0.1-25 mg, or in another embodiment, 0.1-50 mg, orin another embodiment, 0.3-15 mg, or in another embodiment, 0.3-30 mg,or in another embodiment, 0.5-25 mg, or in another embodiment, 0.5-50mg, or in another embodiment, 0.75-15 mg, or in another embodiment,0.75-60 mg, or in another embodiment, 1-5 mg, or in another embodiment,1-20 mg, or in another embodiment, 3-15 mg, or in another embodiment,1-30 mg, or in another embodiment, 30-50 mg, or in another embodiment,30-75 mg, or in another embodiment, 100-2000 mg. In some embodiments,the compounds of this invention may be administered at differentdosages, as a function of time, or disease/symptom/condition severity,or age, or other factors, as will be appreciated by one skilled in theart.

The compounds of this invention may be administered at various dosages.In one embodiment, the compounds of this invention are administered at adosage of 1 mg. In another embodiment the compounds of this inventionare administered at a dosage of 5 mg, or in another embodiment, 3 mg, orin another embodiment 10 mg, or in another embodiment 15 mg, or inanother embodiment 20 mg, or in another embodiment 25 mg, or in anotherembodiment 30 mg, or in another embodiment 35 mg, or in anotherembodiment 40 mg, or in another embodiment 45 mg, or in anotherembodiment 50 mg, or in another embodiment 55 mg, or in anotherembodiment 60 mg, or in another embodiment 65 mg, or in anotherembodiment 70 mg, or in another embodiment 75 mg, or in anotherembodiment 80 mg, or in another embodiment 85 mg, or in anotherembodiment 90 mg, or in another embodiment 95 mg or in anotherembodiment 100 mg.

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more other compound, and/or in combination with other agents usedin the treatment and/or prevention of the diseases, disorders and/orconditions, as will be understood by one skilled in the art. In anotherembodiment, the compounds of the present invention can be administeredsequentially with one or more such agents to provide sustainedtherapeutic and prophylactic effects. In another embodiment, thecompounds may be administered via different routes, at different times,or a combination thereof.

In addition, the compounds of the present invention can be used, eithersingly or in combination, in combination with other modalities forpreventing or treating conditions, diseases or disorders. In someembodiments, such other treatment modalities may include withoutlimitation, surgery, radiation, hormone supplementation, dietregulation, wound debridement, etc., as will be appropriate for thecondition being treated. These can be performed sequentially (e.g.,treatment with a compound of the invention following surgery orradiation) or in combination (e.g., in addition to a diet regimen).

The additional active agents may generally be employed in therapeuticamounts as indicated in the PHYSICIANS' DESK REFERENCE (PDR) 53rdEdition (1999), or such therapeutically useful amounts as would be knownto one of ordinary skill in the art. The compounds of the invention andthe other therapeutically active agents can be administered at therecommended maximum clinical dosage or at lower doses. Dosage levels ofthe active compounds in the compositions of the invention may be variedto obtain a desired therapeutic response depending on the route ofadministration, severity of the disease and the response of the patient.The combination can be administered as separate compositions or as asingle dosage form containing both agents. When administered as acombination, the therapeutic agents can be formulated as separatecompositions that are given at the same time or different times, or thetherapeutic agents can be given as a single composition.

The pharmaceutical composition can comprise the compounds of thisinvention alone or can further include a pharmaceutically acceptablecarrier and can be in solid or liquid form such as tablets, powders,capsules, pellets, solutions, suspensions, elixirs, emulsions, gels,creams, or suppositories, including rectal and urethral suppositories.Pharmaceutically acceptable carriers include gums, starches, sugars,cellulose materials, and mixtures thereof. The pharmaceuticalpreparation containing the compounds of this invention can beadministered to a subject by, for example, subcutaneous implantation ofa pellet; in a further embodiment, the pellet provides for controlledrelease of the compounds of this invention over a period of time. Thepreparation can also be administered by intravenous, intraarterial, orintramuscular injection of a liquid preparation, oral administration ofa liquid or solid preparation, or by topical application. Administrationcan also be accomplished by use of a rectal suppository or a urethralsuppository. The pharmaceutical composition can also be a parenteralformulation; in one embodiment, the formulation comprises a liposomethat includes a complex of a compound of this invention.

The pharmaceutical composition of the invention can be prepared by knowndissolving, mixing, granulating, or tablet-forming processes. For oraladministration, the compounds of this invention or their physiologicallytolerated derivatives such as salts, esters, N-oxides, and the like aremixed with additives customary for this purpose, such as vehicles,stabilizers, or inert diluents, and converted by customary methods intoa suitable form for administration, such as tablets, coated tablets,hard or soft gelatin capsules, aqueous, alcoholic or oily solutions.Examples of suitable inert vehicles are conventional tablet bases suchas lactose, sucrose, or cornstarch in combination with binders likeacacia, cornstarch, gelatin, or with disintegrating agents such ascornstarch, potato starch, alginic acid, or with a lubricant such asstearic acid or magnesium stearate. Examples of suitable oily vehiclesor solvents are vegetable or animal oils such as sunflower oil orfish-liver oil. Preparations can be effected both as dry and as wetgranules. For parenteral administration (subcutaneous, intravenous,intraarterial, or intramuscular injection), the compounds of thisinvention or their physiologically tolerated derivatives such as salts,esters, N-oxides, and the like are converted into a solution,suspension, or emulsion, if desired with the substances customary andsuitable for this purpose, for example, solubilizers or otherauxiliaries. Examples are: sterile liquids such as water and oils, withor without the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solutions, and glycols such as propylene glycols or polyethyleneglycol are preferred liquid carriers, particularly for injectablesolutions.

The preparation of pharmaceutical compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as an aerosol of the polypeptide delivered to thenasopharynx or as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, or pH buffering agents which enhance theeffectiveness of the active ingredient.

For topical administration to body surfaces using, for example, creams,gels, drops, and the like, the compounds of this invention or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In one embodiment, the present invention provides combined preparations.In one embodiment, the term “a combined preparation” defines especiallya “kit of parts” in the sense that the combination partners as definedabove can be dosed independently or by use of different fixedcombinations with distinguished amounts of the combination partnersi.e., simultaneously, concurrently, separately or sequentially. In someembodiments, the parts of the kit of parts can then, e.g., beadministered simultaneously or chronologically staggered, that is atdifferent time points and with equal or different time intervals for anypart of the kit of parts. The ratio of the total amounts of thecombination partners, in some embodiments, can be administered in thecombined preparation. In one embodiment, the combined preparation can bevaried, e.g., in order to cope with the needs of a patient subpopulationto be treated or the needs of the single patient which different needscan be due to a particular disease, severity of a disease, age, sex, orbody weight as can be readily made by a person skilled in the art.

It is to be understood that this invention is directed to compositionsand combined therapies as described herein, for any disease, disorder orcondition, as appropriate, as will be appreciated by one skilled in theart. Certain applications of such compositions and combined therapieshave been described hereinabove, for specific diseases, disorders andconditions, representing embodiments of this invention, and methods oftreating such diseases, disorders and conditions in a subject byadministering a compound as herein described, alone or as part of thecombined therapy or using the compositions of this invention representadditional embodiments of this invention.

Treatment of Conditions or Diseases

In some embodiments, this invention provides compounds, andpharmaceutical compositions comprising the same for the treatment ofconditions and/or diseases specifically involving gonadal cells ortissue, or cells or tissue associated with fertility in a subject.

In some aspects, such conditions/diseases, etc. are effected via theemploy of compounds, which interact with the telomerase enzyme andstimulate and/or increase telomerase expression and/or activity in thetissues and cells of a subject, which in some aspects is via a canonicaland in some embodiments, via a non-canonical pathway.

In some embodiment, such activity is decreased or absent, or in someembodiments, is damaged due to, for example, a disease, disorder orcondition in the subject, and/or arising as a result of a treatment of adisease or disorder or condition in the subject.

In some aspects, the subjects helped by the compounds, compositions andmethods of this invention may be afflicted or predisposed to a caner orprecancerous condition. In some aspects, the subjects helped by thecompounds, compositions and methods of this invention may be exposed toradiation or other toxic therapy.

In some embodiments, the subjects helped by the compounds, compositionsand methods of this invention may be suffering from or predisposed to orhave suffered or been subjected to a) luteal phase defect; b) prematureovarian failure (primary ovarian insufficiency or hypergonadotropichypogonadism); c) impaired sperm production; d) impaired sperm delivery;while sustaining said subject in good health and/or other clinicaltherapeutic and/or diagnostic areas, including any embodiment of what isencompassed by the term “treating” as described herein.

In some embodiments, the subjects helped by the compounds, compositionsand methods of this invention may require or be positively affected byimproved fertility or improved fertility potential in situ or via an invitro protocol, or a process for preparation for a fertility promotingprocedure in situ or in vitro.

For example, and in some embodiments, the subject is male and thecompounds, compositions and methods of this invention expand the spermin or sperm sample taken from a subject for treating impaired or reducedfertility in the subject. In some aspects, the subject is undergoing aGIF, AIF or IVF protocol. In other embodiments, the compounds,compositions and methods of this invention result in enhancedquantity/quality of sperm in the subject without need for any additionalfertility treatment or manipulation. In some embodiments, the subjectmay have been exposed to radiation.

For example, and in some embodiments, the subject is female and thecompounds, compositions and methods of this invention expand the maturefollicles, or ovum in or taken/retrieved from a subject for treatingimpaired or reduced fertility in the subject. In some aspects, thesubject is participating in a GIF, AIF or IVF protocol. In otherembodiments, the compounds, compositions and methods of this inventionresult in enhanced quantity/quality of mature follicles/ovum in thesubject without need for any additional fertility treatment ormanipulation. In some embodiments, the subject may have been exposed toradiation.

In some embodiments, the subjects helped by the compounds, compositionsand methods of this invention may require or be positively affected byexposure of a gonadal or fertility-associated cell or tissue beingisolated from or within a subject having a cancerous or precancerouscondition, suffering from an endocrine disorder that negatively impactssteroidogenesis, suffering from infertility or predisposed toinfertility, or suffering from a genetic disorder or other disordercausing premature failure of a gonadal cell or tissue.

In one embodiment, the compounds and compositions of this inventionactivate telomerase, and methods as described herein are useful thereby.

Example 3 as further described herein demonstrated that the compounds ofthis invention increased telomerase expression and activity in the ovaryat all estrous stages evaluated, even when using a single dose of theembodied compounds.

Similarly, the embodied compounds of this invention facilitated/promotedincreased ovarian size and weight at each stage of estrous cycle andaccelerated the estrous cycle (FIG. 12).

The embodied compounds promoted and enhanced ovulation, (FIG. 13).

The embodied compounds induced secretion of progesterone, as well. Thecompounds of this invention increased the number of embryos.

The embodied compounds thus clearly improve and promote female fertilityand contribute greatly to increasing fertility therapeutic strategies,and in some aspects are particularly suitable in supporting IVFtreatments, in consideration of the data provided in the next example,as well.

Example 4 demonstrated that the embodied compounds are also useful in,inter alia, protecting male gonadal cells/tissue from X-ray inducedmorphological damage (FIG. 22).

A beneficial effect of the embodied compounds on sperm count atdifferent times after X-ray radiation was also demonstrated (FIG. 25).While the sperm counts from X-ray treated subjects were significantlyreduced, subjects exposed to X-ray irradiation but administered theembodied compounds showed significantly increased numbers of sperms inthe epididymis (FIG. 25).

The embodied compounds of this invention are therefore, inter alia,useful in promoting fertiliyt in subjects exposed to radiation, andthereby in some aspects, significantly restoring tissue morphology,increased CREM expression and decreased DSB formation. Furthermore,treatment with select compounds, such as Compound 68 increased the spermcount in the epididymis, at different time points post radiation,indicating its promise as a therapy to restore male fertility inirradiated subjects.

Such protective effects in gonadal tissue following irradiation werealso demonstrated effective in female subjects (FIG. 26).

The ability of a compound to increase telomerase expression and/oractivity in a cell can be determined using the TRAP (Telomeric RepeatAmplification Protocol) assay, which is known in the art (e.g., Kim etal., U.S. Pat. No. 5,629,154; Harley et al., U.S. Pat. No. 5,891,639).The activity is typically compared to the activity similarly measured ina control assay of such cells (e.g., a telomerase activity 50% greaterthan observed in a solvent control). Cell lines suitable for use in theassay, may comprise normal human fibroblasts (Now) or normal humankeratinocytes (NHK).

In some embodiments, the telomere length may serve as useful indicatorfor the telomerase expression and/or activity. In one embodiment,telomerase expression and/or activation is important in treating cancer,premature aging syndrome or segmental progeria, genetic anomalies andage-related diseases. The telomere length has distinct patterns ofexpression in specific disease progression, and is a function of itsactivation, thus measuring such length as a function of treatment hasvalue, in some embodiments, in terms of the prognosis of differentdiseases.

In one embodiment, telomere length can be measured by Southern blot,hybridization protection assay, fluorescence in situ hybridization, flowcytometry, primed in situ, quantitative-polymerase chain reaction andsingle telomere length analysis, which are all techniques known in theart (Kah-Wai Lin and Ju Yan, J. Cell. Mol. Med., 2005, Vol 9, No. 4,977-989).

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES Example 1 Synthesis of Compounds

Compounds of the invention were synthesized as described in PCTInternational Application Numbers WO 2008/149,345; WO 2008/149353; WO2008/149,346, all of which are fully incorporated herein by reference intheir entirety.

In particular, the following compounds were utilized:

Synthetic methods for preparing compound 70, may include the following:

1,1,1-tris(4-methoxyphenyl)-ethane—To a solution of 1.53 g, 5 mM,1,1,1-tris(4-hydroxyphenyl)-ethane in 20 ml ethanol and 10 ml water wereadded, during 1 hour and simultaneously in portions, 1 g, 25 mM, NaOH in10 ml water and 5.1 gr, 40 mM of dimethyl sulphate (1:8 molar ratio).The solution was then refluxed for 1 hour, and stirred 70 hours at RT.The white precipitate was filtered, washed with water and dried to give1.74 g. Recrystalization twice from 50 ml ethanol gave 1.15 gr whitecrystals, 66% yield, m.p.-160°. TLC Rf=0.85 in CH₂Cl₂.

NMR CDCl₃ δ 6.99, 6.79 (12H, AB_(q), J_(AB)=8.8 Hz), 3.78 (9H, s, OCH₃),2.11 (3H, s, CH₃).

To a solution of 0.49 gr, 1.4 mM, 1,1,1-tris(4-methoxyphenyl)-ethane,from A, in 22 ml 1,2-dichloroethane were added in portions 1.65 gr, 10.2mM, (7.3:1 ratio) of bromine in 5 ml 1,2-dichloroethane. The solutionwas stirred at RT overnight and heated for 3 hours to 700, and workedup(sodium thiosulphate) to give 1.0 gr crude product. TLC shows no SM, butNMR is mixture (m at 6.90 ppm, and 4 methoxy). The solid was brominatedagain with 1 gr bromine, reflux 18 hours. Workup and Trituration withhot ethanol gave 0.27 gr white solid, 23% yield, mp −160°. TLC Rf=0.95in CH₂Cl₂.

NMR CDCl₃ δ 7.16 (6H, s, ArH), 3.92, 3.91 (6:4 ratio) (9H, 2s, OCH₃),2.04, 2.03 (4:6 ratio) (3H, s, CH₃).

Example 2 The Effect of Embodied Compounds of this Invention onSteroidogenesis in Rat Granulosa Cells Materials and Methods

Immortalized rat granulosa cells expressing FSH receptor were treatedwith either pregnant mare serum gonadotropin (PMSG), or the indicatedcompounds, or both for 6 hours. Protein extracts prepared from thesecells served for telomerase activity measurement using a telomere repeatamplification protocol (TRAP) assay.

Briefly, protein extract incubated with oligonucleotide, which serves asa template for telomerase (TS), for 45 minutes at 30° C. followed by PCRwith reverse primer (ACX) and internal control. PCR products wereseparated on 4.5% agarose gel. RNA extracts were used for cDNA synthesisand quantitative real time PCR with specific primers to Telomerasereverse transcriptase (TERT), Steroidogenic acute regulatory protein(StAr) and β-actin as a reference gene. To measure progesterone levelssecreted by the cells, granulosa cells were cultured with or without 1IU/mL PMSG and DMSO, Compound 79 50 nM or compound 70 50 nM in thepresence of 100 nM dexamethasone. After 24 hours progesterone levelscell-culture medium were determined by radiommunoassay (RIA). For cellproliferation measurement, 1×10⁵ cells were seeded in a 96 wells platewith 1 IU/mL PMSG and DMSO, Compound 79 50 nM or Compound 70 50 nM.After 24 hours cells viability was measured by XTT proliferation assay.

Preparation of Proteins Extract

The ovaries were homogenized and centrifuged at 1200 RPM at 4° C. for 10min, then suspended in CHAPS buffer (10 mM Tris-HCl pH-7.0, ImM MgCl2,ImM EDTA pH-0.5, 0.1 mM PMSF, 0.5% 3[(3 Cholamidopropyl)dimethylammonio]-propanesulfonic acid [CHAPS] and 10% glycerol) on icefor 30 min and centrifuged again at 13500 RPM at 4° C. for 30 min, thesupernatant was collected. Total proteins concentration was determinedusing the Bio-Rad protein assay kit (Bio-Rad Laboratories, Germany).

Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase activity in mice ovary was evaluated by a slight modificationof the TRAP assay [Grin, Y., T. Admoni, and E. Priel, Telomeraseactivity in the various regions of mouse brain: non-radioactivetelomerase repeat amplification protocol (TRAP) assay. J Vis Exp,2014(91): p. e51865]. The assay contains three main stages: Telomerasereaction, PCR reaction and b high resolution agarose mini-gelelectrophoresis analysis. Proteins extract (0.5 μg/μl) was incubatedwith 1 μl of 10×TRAP assay reaction mix: (20 mM Tris-HCl pH 8.2, 63 mMKCl, 1.5 mM MgCl2, 1 mM EDTA, 0.1 mg/mL BSA and 0.05% Tween 20) and

0.1 μg telomerase substrate (TS) primer (5′-AATCCGTCGAGCAGAGTT-3′, 0.1μg/μl), 2.5 mM dNTP's and UPW to a final volume of 10 μl for 45 minutesat 30° C. water bath. Then, the telomerase reaction products wereamplified by PCR using reaction mix containing 0.1 μg ACX primer

(5′-GCGCGGCTTACCCTTACCCTTACCCTAACC-3′, 0.2 μg/μl), 1.25 μl Titanium Taqpolymerase buffer ×10 and 0.25 μl Titanium Taq-polymerase ×50. Internalstandard primer used as a control: 0.5×10-15 μg IS primer (5′AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3′, 1×10-15 μg/μl) and 0.025 μg ISRprimer (5′-ATCGCTTCTCGGCCTTTT-3′, 0.05 μg/μl). 34 PCR cycles aspreviously described [Grin, Y., T. Admoni, and E. Priel, Telomeraseactivity in the various regions of mouse brain: non-radioactivetelomerase repeat amplification protocol (TRAP) assay. J Vis Exp,2014(91): p. e51865] were used for the detection of the products. Theseproducts were separated on a 4.5% high-resolution agarose mini-gel anddetected with Gel-Red solution (×10000 stock solution diluted to ×3working solution with 0.01M NaCl) for 20 minutes. The gels were filmedusing a UV trans-illuminator digital camera system at 302 nm wavelength.All assays included CHAPS buffer as negative control and positivecontrol—proteins extract from Glioblastoma cells.

Quantification of Telomerase Activity

TRAP assay products were quantified by densitometric analysis using theEZQuant software (EZQuant Ltd. Rehovot, Israel) and normalized to IS(Internal standard). We also used positive control (proteins extractfrom cancer cells) in each of the experiments as a constant referencefor the comparison between the different experiments. The data whichwere normalized to the IS were calculated as % from the positive controlvalues in each experiment.

Isolation of Total RNA and cDNA Preparation

Mouse ovary was removed and homogenized in Tri-reagent RNA extractionbuffer (Sigma-Aldrich, Rehovot, Israel) and RNA was isolated accordingto the manufacture's protocol. RNA concentration was determined usingNanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc.,Pittsburgh Pa., USA). The RNA (1000 ng) was transcribed to cDNA with the“Revert Aid First Strand cDNA Synthesis Kit” (Thermo Fisher ScientificInc, USA) according to the manufacturer's instructions.

Real-Time PCR

RT-PCR was used to measure gene expression of mTERT which was normalizedto the expression of the housekeeping gene: β-actin. cDNA (25 ng) wasadded to the reaction mixture which includes: mixture of primers (FW andRV), SYBR-Green and UPW. The primers used for PCR amplification of mTERTwere: FW 5′-GAAAGTAGAGGATTGCCACTGGC-3′ and

RV 5′-CGTATGTGTCCATCAGCCAGAAC-3′. The primers for β-actin were: FW

5′-GATGTATGAAGGCTTTGGTC-3′ and RV 5′-TGTGCACTTTTATTGGTGTG-3′.

Products were measured by ΔΔCt method in which mTERT gene is calculaterelatively to β-actin.

Progesterone Levels in Mouse Serum

Blood samples were taken from the sacrificed mouse and followingcentrifugation (3800 rpm, 10 min) the serum was sent to the clinicalendocrinology laboratory (Soroka University Medical Center, Beer-Sheva,Israel) for determination of progesterone levels.

Results

Gonadotropin stimulation induces TERT expression and telomeraseactivity. FIG. 1 graphically plots the relative TERT Expression (%) as afunction of increasing PMSG concentration. FIG. 2 shows the proteinlevels from a TRAP assay of granulosa cells treated with 10 IU/mL PMSGfor 6 hours.

The compounds increases TERT expression and telomerase activity (SeeFIGS. 3 and 4). Granulosa cells treated with two compounds for use inaccordance with this invention (compounds 79 and 70), at 50 nM and 200nM for 6 hours, respectively, when assayed for protein expression viaTRAP and TERT relative mRNA levels via qRT-PCR support increasedexpression in a dose-dependent fashion. Suprisingly, Compound 70 furtherincreased telomerase activity and expression in granulosa cells ascompared to compound 79 (FIG. 3).

Increasing TERT expression by the embodied compounds induced theexpression of Steroidogenic acute regulatory protein (StAr). FIG. 5shows granulosa cells treated with compounds 79 and 70, at 50 nM for 6hours demonstrate increased expression of StAr.

Increasing TERT expression by the embodied compounds increasesprogesterone levels specifically produced by granulosa cells aftergonadotropin stimulation, while no significant effect on cell growth wasdetected. FIGS. 6 and 7 demonstrate

FIG. 6 demonstrates Progesterone levels in Granulosa cells treated withcompound 79 or compound 70, at 50 nM with or without PMSG 1 IU/mL for 24hours as determined by radioimmunoassay (RIA). FIG. 7 demonstratesgranulosa cell viability following treatment with the compounds 79 and70, 50 nM with 1 IU/mL PMSG for 24 hours, as measured by XTTproliferation assay.

Taken together, it is demonstrated herein for the first time, thatgonadotropin (FSH) stimulation in granulosa cells increased telomeraseexpression, which in turn contributes to the pathway by whichFSH-controlled granulosa cells proliferate.

Increasing telomerase (by the administered compounds) in granulosacells, affected the expression of genes involved in the stimulationpathway of FSH and the progesterone levels secreted by the cells,supporting the involvement of telomerase in steroidogenesis in granulosacells and the ability to positively affect female fertility thereby.

Example 3 The Effect of Embodied Compounds of this Invention onOvulation In Vivo Materials and Methods

Female ICR mice at least 3 months old were administered embodiedcompounds of this invention (Compound 79 and Compound 68) (6 mg/Kg) orDMSO alone (0.5%) by subcutaneous administration. Vaginal smears weretaken at baseline and at 12 hours following the administration todetermine estrous cycle stage and mice were then sacrificed.

The ovaries were weighted; one was taken for protein extraction formeasuring telomerase activity by TRAP assay, and the other one for thehistological examination using hematoxylin-eosin staining of the ovaryand its follicles.

Histological Analysis

The mice ovaries were removed and fixed with 4% paraformaldehydeovernight, dehydrated in increasing ethanol concentrations to xylene,and embedded in paraffin. A rotary microtome was used to produce 5 msections, which were mounted on Superfrost plus slides. The sectionswere dried and heated at 37° C. overnight and stored in dry place.Paraffin-embedded sections were deparaffinized in xylene and rehydratedin decreasing concentrations of ethanol. Then the sections stained withhematoxylin-eosin or subjected to immunochemical and Immunofluorescentassays [Liani-Leibson, K., I. Har-Vardi, and E. Priel, Inhibition oftopoisomerase I by anti-cancer drug altered the endometrial cyclicityand receptivity. Curr Mol Med, 2014. 14(1): p. 141-50].

Immunofluorescence

All sections were initially treated with Antigen retrieval solution; thesections were heated for 5 min in 10 mM sodium citrate solution pH=6.Nonspecific binding was blocked by incubating the sections with 3% BSA.Monoclonal mouse anti-PCNA, clone PC 10 sc-56 (Santa cruz Dallas, Tex.USA) diluted 1:200 in PBS+3% BSA was used as the primary antibody.Sections were incubated with primary antibody overnight at 4° C. Thenext day, sections were washed with PBS and PBS contains 0.1% TWEEN 20(Sigma) The secondary anti mouse antibody (cy3) (Jackson Pa., USA) wasapplied for 2 hours at room temperature. The sections were washed withPBS and PBS contains 0.1% TWEEN 20 and DAPI (Sigma) was applied for 10min. Finally, sections were washed, and covered by water-based mountingmedium. PCNA labeling was examined using Pannoramic midi (3D Histech)device.

Pairings Between Male and Female Mice

Female ICR mice were treated with a single s.c. injection of Compound79, Compound 68 and Compound 70 6 mg/kg or with DMSO 0.5%. Immediatelyafter the injection the female mouse was put into a cage of the malemouse, after 14 days female mouse was sacrificed and the number ofembryos in the uterus were counted.

Other methods were as described hereinabove in Example 2.

Results

Telomerase activity peaks at the age of 18 days in ovary of femalemouse. Telomerase activity in protein extracts derived from ovaryobtained from mice at various ages: 10 days, 18 days, 1 month and 3months (n=12, 3 per group) was determined by modification of the TRAPassay [Grin, Y., T. Admoni, and E. Priel, Telomerase activity in thevarious regions of mouse brain: non-radioactive telomerase repeatamplification protocol (TRAP) assay. J Vis Exp, 2014(91): p. e51865] andquantified by densitometric analysis using the EZquant software aspreviously described [Eitan, E., et al., Novel telomerase-increasingcompound in mouse brain delays the onset of amyotrophic lateralsclerosis. EMBO Mol Med, 2012. 4(4): p. 313-29; Tichon, A., et al.,Oxidative stress protection by novel telomerase activators inmesenchymal stem cells derived from healthy and diseased individuals.Curr Mol Med, 2013. 13(6): p. 1010-22; Tichon, A., et al., Telomeraseactivity and expression in adult human mesenchymal stem cells derivedfrom amyotrophic lateral sclerosis individuals. Cytotherapy, 2009.11(7): p. 837-48]. The results depicted in FIG. 8 show that telomeraseactivity in the ovary declines with age, and the peak of the activitywas observed at 18 days old. To examine whether the level of telomeraseexpression is influenced by the stage of estrous cycle in the mouseovary, we determined the stage of the estrous cycle of the female mouse(3 months old), prior to sacrificing, using the common vaginal smearsmethod. Total RNA was extracted from the ovaries, cDNA was prepared andReal-Time PCR was performed with the appropriate mTERT primers. Theresults in FIG. 9 show that the expression of TERT in mouse ovary didnot significantly changed through the estrous cycle

Administration of embodied compounds increased telomerase activity inthe mouse ovary at various ages. Mice of different ages were assigned togroups (10 days, 18 days, 1 month and 3 months) and treated withCompound 79, Compound 68, DMSO as a vehicle control and untreated (n=48,3 mice per group). Compound 79, and Compound 68 (6 mg/Kg) or DMSO (0.5%)were injected subcutaneously in mice and after 12 hours the mice weresacrificed, the ovaries were removed, and proteins extract used formeasuring telomerase activity by TRAP assay. A single dose of Compound68 significantly increased telomerase activity (up to 1.5 fold) at allthe examined ages (FIG. 10 A-E) while Compound 79 significantlyincreased telomerase activity only in the ovary of 3 months old mouse(10E).

The compounds of this invention increase telomerase expression andactivity in mouse ovary at all estrous stages. To determine if theincrease of telomerase expression and activity by the compounds dependson the estrous stages we treated mice with a single dose of theindicated compounds at the various Estrous stages as described above.Twelve hours post treatments the mice were sacrificed, the ovaries wereremoved: one was taken for protein extraction for measuring telomeraseactivity by TRAP assay, and the other one for RNA extraction formeasuring the level of TERT mRNA expression using qRT-PCR. In mouseovary at proestrus, estrus and diestrus stages, the compoundssignificantly increased telomerase expression (up to 1.5 fold) (FIG.11A) and telomerase activity (11B). When the compounds were administeredat the metestrus stage they increase telomerase expression and activitybut not significantly.

Activation of telomerase increased ovarian size and weight at each stageof estrous cycle and accelerate the estrous cycle. The ovaries of themice from the above described experiments were weighted prior to thevarious analysis. We observed an increase (of up to 40%) in mouseovary's size and weight following treatment with the indicated compoundscompared to untreated and DMSO, at all the estrous cycle stages (FIG.12). To examine the biological effects of increasing telomerase in theovary by the compounds on the estrous cycle, mice (3-5 months old) wereuntreated or treated with the compounds (a single dose of Compound 79,Compound 68) or the vehicle (0.5% DMSO) at the various stages of theestrous cycle, and 12 hrs after treatment the stage of the Estrous cyclewas determined by the component of cells in the Vaginal smears. Theresults presented in FIG. 13 show that mice injected with a single doseof the compounds in the proestrus (P) or estrus (E) stages were found tob, 12 hrs post treatment, in the metestrus stage. While the controluntreated or vehicle treated mice were after 12 hrs at the estrus stage,as expected. No changes in the estrous cycle stage, 12 hrs posttreatment, were observed when the compounds were injected at thediestrus or metestrus stages.

The embodied compounds promote and enhance ovulation, as evidenced bychanges in vaginal smears at proestrus and estrus stages before andafter 12 h of treatment (FIG. 13). Vaginal smears were taken from femalemice following by s.c. injection with DMSO, Compound 79 and Compound 68,as indicated. (A). Before treatment proestrus stages (A), estrus stages(C). After 12 hours of treatment the vaginal smears were taken again.proestrus stages (B), estrus stages (D). The vaginal smears were stainedby crystal violet.

Histological analysis of the ovaries derived from 3 month old micetreated with the compounds as described extended these findings. Themice were divided to groups according to their estrous cycle stages (P,E, M, D). At each stage of the estrous cycle mice were untreated ortreated with a single dose (6 mg/Kg) of Compound 70, 79, Compound 68, orDMSO 0.5%). Twelve hours post treatment the mice were sacrificed, theovaries were removed for the histological examination usinghematoxylin-eosin staining. As can be seen in FIG. 14, treatment of miceat the pro-estrus (P) and estrus (E) stages with Compound 70, 79 orCompound 68 caused, 12 hrs after treatment, more corpus luteum (CL) thanin the control group (UT, DMSO) suggesting an increase of ovulation(FIG. 14A, 14B).

Treatments at met-estrus (M) stage demonstrate, 12 hours post treatment,many corpus luteum (CL) in all of the treatment groups, but in Compound70, 79 or Compound 68 treated mice more developed follicles (whitearrow) and plurality of layers of granulosa cells (GC) around theoocytes were seen; while in the control groups mostly primordialfollicles (blue arrow) were detected (FIG. 14C). Treatments at theDi-estrus (D) stage with Compound 68 shows 12 hours post treatment, aplurality of corpus luteum (CL) and Compound 79 shows multiple layers ofgranulosa cells (GC) around the oocytes (FIG. 14D).

Examination of cells proliferation in the ovary following the varioustreatments was performed using anti-PCNA antibody, a known proliferationmarker. The results revealed that untreated or vehicle treated mice atthe estrus stage demonstrated significant PCNA staining in the GC of thevarious follicles (FIG. 15A), while following treatment the appearanceof CL with less PCNA staining of GC was detected, which is compatiblewith the met-estrus stage of the ovary (FIG. 15A). When the mice at themet-estrus stage were treated with the embodied compounds (FIG. 15B) theamount of GC in the follicles that demonstrate PCNA staining increasedcompared to untreated or vehicle treated mice suggesting the activationof follicles by the compounds.

The compounds of this invention induced secretion of progesterone. Todetermine if the increase in the number of CL observed after treatmentwith the compounds produced functional corpus luteum that secretesprogesterone, blood samples were taken 12 hours post treatments from thesacrificed mouse and the serum was analyzed for progesterone levels. Theresults showed that 12 hrs post treatment a significant increase (up to5 folds compared to DMSO or UT) in the secretion of progesterone in micethat were treated with Compound 79 at P, E, and M stages of the estrouscycle was observed (FIG. 16). Treatment with Compound 80 demonstrated anincrease in progesterone only when it was administered at the met-estrusand pro-estrus stages. These data are compatible with the abovedescribed results which demonstrate that differential treatment andgeneral treatment with the indicated compounds at pro-estrus and estrusstages accelerates the appearance of the met-estrus stage that accordingto the literature shows a high secretion of progesterone.

The compounds of this invention increase the number of embryos. Thehistological examination of ovarian tissue showed a high numbers ofcorpus luteum following treatment with the embodied compounds,relatively to untreated mouse at the same estrous cycle stage, whichindicates increase in the number of ovulated oocytes. To examine if theoocytes ovulated following treatments are viable, we tested the numberof embryos generated following the various treatments: Compound 79 orCompound 68, DMSO and untreated, and Compound 70. Following a single s.cinjection of the respective compound (6 mg/kg) or DMSO (0.5%) singlefemale mice were put into a cage containing a male mouse. Fourteen daysafter, the female mice were sacrificed and the number of embryos in theuterus of each treated mouse was determined. The data presented in FIG.17 show that the number of embryos in the treated mice significantlyincreased (up to 50%) compared to the controls (DMSO or UT) mice.

Taken together, this example shows that telomerase activity is presentin mouse ovary, which decreases with age, peaking at the age of 18 days,while the level expression of TERT doesn't seem to be appreciablymodified at each stage of estrous cycle.

In contrast to naïve mice, mice treated with the embodied compounds ofthis invention demonstrated increased telomerase activity at all ages,in particular at the age of 3 months that is considered peak fertilityage in female mice. Increasing telomerase activity shows moreproliferation of granulosa cells and more development of antral andpre-ovulatory follicles, in addition to high ovarian weight and size. Weobserved that the embodied compounds increase telomerase expression andactivity significantly in mouse ovary at all estrus stages but not atmet-estrus, that incretion by the embodied compounds in estrus andpro-estrus stages accelerate the appearance of the met-estrus stageaccording to typical vaginal cells and increase the numbers of corpusluteum follicles at all stages after 12 hours of single treatments. Theembodied compounds also have an effect on secretion of progesterone asin luteal phase, regardless of estrus cycle stages. After the pairingswe observed that the compounds increase the number of embryos,supporting that the compounds accelerate the ovulation process anddeveloped more pre-ovulatory follicles with mature oocytes.

The embodied compounds thus clearly improve and promote female fertilityand contribute greatly to increasing fertility therapeutic strategies,and in some aspects are particularly suitable in supporting IVFtreatments, in consideration of the data provided in the next example,as well.

Example 4 Variations in Telomerase Activities in Granulosa Cells Derivedfrom Women Undergoing IVF Procedures Background, Materials and Methods

Although the telomerase (Telo) enzyme has a catalytic subunit withreverse transcriptase (RT) activity-TERT, which performs de novosynthesis of one strand of the telomeric DNA in its canonic role, thereare known non-canonical roles, as well, which include cellularproliferation, gene expression and regulation, mitochondrial functionlike protection against oxidative stress and apoptosis, signalingpathways, genesis of double-stranded RNAs in order to silencemitochondrial RNAs. Telomerase is repressed in somatic cells of longlived organisms, but detected in human germ cells. The enzyme is anessential nuclear enzymes that exists in all living organisms, in whichit plays a crucial role in genomic stability. Little is known, however,about the enzymesin developing granulosa cells (GCs). Even less isunderstood in terms of the modulation of same in conditions impactinginfertility, such as, for example, in IVF therapy.

To evaluate the role/modulation of the enzyme during IVF treatment, GCswere collected from follicular liquid of women undergoing IVF after theoocyte was taken for IVF procedures. GCs were separated under amicroscope and washed with saline. Examination of telomerase activitywas determined in extracts derived from patient's GCs (WC— whole cellsand DB-DNA-bound fraction) using the TRAP assay. Determination of theexpression of telomerase in the various extracts was obtained byexamination of the level of TERT protein using a specific ELISA kit.

Results

FIGS. 18A and B relate to the relative Telomerase activity in womenundergoing IVF. The telomerase DNA products obtained by the TRAP assay(FIG. 18A—for 0.5 μg protein of WC extract and FIG. 18B—0.1 μg proteinof DB extract), were analyzed by densitometry. Telomerase activity wascalculated as % of the data obtained for a permanent positive control(protein extracts from glioblastoma cells). The results are means±SD ofat least 3 independent TRAP assays.

There is a correlation between high and low blood estrogen levels, andaverage enzymes activities. Telomerase (telo) activity was evaluated inFIG. 19:(A) (WC: p=0.01, DB: p=0.42) n=9 for high, n=12 for low. Theaverage enzyme activity of telo (in WC and DB extracts) was calculatedfor high and low estrogen levels. High estrogen level was determined as≤1500 ng/ml.

Evaluation of IVF patients provided for the finding that there exists acorrelation between an infertility diagnosis and telomerase activity(FIG. 20), with n=9 for female infertility, n=12 for male infertility(p=0.159. Similarly, the relative Telomerase expression in womenundergoing IVF was evaluated (FIG. 21). WC protein extracts wereanalyzed by ELISA kit (EIAab). FIG. 21A—Telomerase expression wascalculated as % of the total protein expression with correlation toblood estrogen levels. FIG. 21B—Telomerase expression of individualwomen.

Taken together, these studies supported that variations exist intelomerase activity in women undergoing IVF (in WC and BD fractions). Anegative correlation exists in terms of enzyme activity as compared toblood estrogen levels. Preliminary analysis assessing the women based ontheir cause of infertility, showed alterations in telomerase activity,where low telo activity was observed within the group of “femaleinfertility factors” and not in the group of “male infertility factors”.These results further support that the embodied compounds would beuseful specifically when treating patients undergoing IVF, as part ofinfertility management.

Example 5 Increasing Telomerase by the Embodied Compounds ProtectedMouse Testes from Damages Induced by X-Ray Radiation Background andMaterials and Methods

Telomerase Enzyme is responsible for the re-elongation of telomeres atthe ends of chromosomes and for providing genome stability. Most somaticcells contain low or undetected expression of telomerase but during thespermatogenesis process telomerase is active and its catalytic subunitTERT is expressed. Factors that altered the spermatogenesis process likeX-ray radiation significantly affect male fertility. It became ofinterest therefore as to whether the embodied compounds of thisinvention, would be able to compensate or otherwise protect the testisfrom the damaging effects of radiation exposure.

Toward this end, ICR mice (3 months old) were injected with 6 mg/kg ofCompound 79, Compound 68 or Compound 70, or mice were subjected to X-rayradiation (2.5 Gy) followed by immediately injection of the respectivecompounds. Control mice were untreated or vehicle treated with orwithout exposure to X-ray radiation. The testes were removed 12 hrsafter treatments and subjected to protein extracts or total RNApreparations for the examination of telomerase activity by TRAP assay,telomerase expression by Western blot and real time PCR. Testes werealso taken for immunohistochemical and immunofluorescence analysis withvarious spermatogenesis markers. Epididymis was also taken for spermcount.

Results

The compounds protected mouse testes from X-ray induced morphologicaldamage. H&E staining shows a significant destruction in the morphologyof the testicular tissue particularly in the spermatogonia cells layer,following irradiation (FIG. 22). Compound 68 treatment, however,protected the spermatogonia cells layer from the damaging effects ofX-ray and the morphology of the testicular tissue remained intact.

There is a beneficial effect of Compound 68 and Compound 70 on CREMexpression in X-ray irradiated testicular tissue. Examination of CREMexpression in the testis before and after X-ray radiation, revealed asignificant alteration in the expression pattern in X-ray treated testiscompared to the untreated mice (FIG. 23). Compound 68 and Compound 70treatments restored the expression of the spermatogenesis markers (FIG.23A and FIG. 23B, respectively).

Compound 70 treatment protected mouse testes from the X-ray radiationdamage, as well (FIG. 24). IF analysis of γ-H2AX (green) marker showed asignificant increase of DSB formation 12 hrs post-irradiation, comparedto UT samples. In addition, treatment with Compound 70 only did notcause DNA damage. In irradiated mice, Compound 70 treatment demonstrateda decrease in the expression levels of γ-H2AX in the various cell types,mainly in spermatogonia, indicating that Compound 70 treatment protectsthe testis tissue from DNA damage induced by X-ray radiation.

Beneficial effect of the embodied compounds on sperm count at differenttimes after X-ray radiation (FIG. 25). Measurement of sperm cells numberfrom the epididymis in non-irradiated mice followed by treatment withthe indicated showed a significant increase 12 hrs. post treatments withCompound 68 and Compound 70, as compared to DMSO. Sperm count from X-raytreated and untreated mice revealed a significant reduction in spermcount, while treatment with Compound 68 and Compound 70 of X-rayirradiated mice significantly increased the numbers of sperms in theepididymis by 1.5-2.7 folds 12 hrs post-radiation (FIGS. 25 A and B).Treatment of irradiated mice with Compound 70 increased sperm count at 9and 30 days post irradiation (FIGS. 25C and D).

The beneficial effect of the embodied compounds on sperm morphology isalso demonstrated at different times after X-ray radiation (FIG. 26).FIG. 26A quantitatively demonstrates the reduction in the number ofdefective sperm cells, based on morphologic evaluation, following X-rayexposure, when cells are treated with Compound 68 or Compound 70. FIG.26B provides a micrograph of sperm morphology as a frame of reference.The embodied compounds therefore not only increase sperm count, but alsoincrease sperm quality.

FIG. 27 demonstrates the positive effect of the embodied compound 70 onsperm count (FIG. 27A) and motility (FIG. 27B) in old mice (16 months).The production of sperm in old mice is relatively low and unlike inyounger mice, it is sensitive to DMSO treatment. However treatment withthe embodied compounds not only overcome the reduction of sperm countinduced by DMSO but also increased sperm production over the basal levelin old mice which indicates the potential of these compounds to increasemale fertility in older subjects.

Taken together, these results support the ability of the embodiedcompounds of this invention in X-ray irradiated testes to significantlyrestore tissue morphology, increase CREM expression and decrease DSBformation. Furthermore, treatment with select compounds, such asCompound 68 increased the sperm count in the epididymis, at differenttime points post radiation, indicating its promise as a therapy torestore male fertility in irradiated subjects. Finally, protectiveeffects in elder mice hold promise for improving fertility in an agingor prematurely aging population.

The protective effect in gonadal tissue following irradiation is alsoseen in female subjects. FIG. 28 depicts an H&E stained ovarian sectionof X-ray irradiated tissue, where Compound 70 treatment followingirradiation clearly rescues ovarian tissue exposed to radiation.

The embodied compounds also modulate sex hormone production (FIG. 29).Expression of the gonadotropins LH and FSH was assessed in cells derivedfrom murine pituitary gland. Compound 79 increased expression of LHabove untreated levels at 6 hours post treatment, and decreased FSHlevels at 12 hours, while compounds 68 and 70 increased LH productionabove untreated levels at 12 hours and decreased FSH expression relativeto untreated samples at 6 hours, as assessed by RT-PCR.

FIG. 29 demonstrates the effect of compound 534 on the expression ofgonadotropins: LH and FSH in a pituitary gland cell line. FIG. 30demonstrates the results of expression of LH and FSH 12 hours aftertreatment with compound 534 and control animals, where there is acorrelation between the telomerase expression and the gonadotropinexpression, in control and treated mice. Animals in pro-estrus (P) showthe highest expression of telomerase (FIG. 30A), and concomitantly highlevels of LH (FIG. 30C) and FSH (FIG. 30E), and progression throughestrus and met-estrus, similarly LH expression follows that oftelomerase expression and the trend appears with respect to FSH, aswell. In animals treated with compound-534, 12 hours after treatment, itwould seem that more animals went through estrus (E) and met-estrus (M),i.e. the compound seems to accelerate the cycle, as noted above (CompareFIGS. 30B versus 30A, 30D versus 30C and 30F versus 30E).

The relative modulation of expression of these gonadotropins plays animportant role in both male and female fertility, supporting an effectof the compounds activity, as well, in modulation of gonadotropinexpression.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

What is claimed is:
 1. A method of promoting, improving, recovering orrestoring fertility in a subject in need thereof, comprising contactinga gonadal or fertility-associated cell or tissue with a compoundrepresented by the structure of formula I:

wherein Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and R₁₀ is nothing, H, D, OH, halogen, oxo, nitro,CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone,—COOH, ester, trifluoromethyl, amide, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl,alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceuticallyacceptable salt, pharmaceutical product, hydrate, N-oxide, crystal orany combination thereof.
 2. The method of claim 1, wherein promoting,improving, recovering or restoring fertility in a subject beneficiallymodulates gonadotropin expression or steroid hormone expression in termsof timing or quantity or a combination thereof.
 3. The method of claim1, wherein promoting, improving, recovering or restoring fertility in asubject improves follicle maturation in terms of timing or quantity or acombination thereof.
 4. The method of claim 1, wherein promoting,improving, recovering or restoring fertility in a subject improves spermquantity, quality, motility or a combination thereof.
 5. Use of acompound represented by the structure of formula I:

wherein Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and R₁₀ is nothing, H, D, OH, halogen, oxo, nitro,CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone,—COOH, ester, trifluoromethyl, amide, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl,alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceuticallyacceptable salt, pharmaceutical product, hydrate, N-oxide, crystal orany combination thereof for promoting, improving, recovering orrestoring fertility in a subject in need thereof.
 6. The use of claim 5,wherein promoting, improving, recovering or restoring fertility in asubject beneficially modulates gonadotropin expression or steroidhormone expression in terms of timing or quantity or a combinationthereof.
 7. The use of claim 5, wherein promoting, improving, recoveringor restoring fertility in a subject improves follicle maturation interms of timing or quantity or a combination thereof.
 8. The use ofclaim 5, wherein promoting, improving, recovering or restoring fertilityin a subject improves sperm quantity, quality, motility or a combinationthereof.
 9. A method of promoting, improving, recovering or restoringfunction to gonadal cells or tissues in a subject in need thereof,comprising contacting a gonadal or fertility-associated cell or tissuewith a compound represented by the structure of formula I:

wherein Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and R₁₀ is nothing, H, D, OH, halogen, oxo, nitro,CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone,—COOH, ester, trifluoromethyl, amide, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl,alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceuticallyacceptable salt, pharmaceutical product, hydrate, N-oxide, crystal orany combination thereof.
 10. Use of a compound represented by thestructure of formula I:

wherein Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and R₁₀ is nothing, H, D, OH, halogen, oxo, nitro,CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone,—COOH, ester, trifluoromethyl, amide, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl,alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceuticallyacceptable salt, pharmaceutical product, hydrate, N-oxide, crystal orany combination thereof for promoting, improving, recovering orrestoring function to gonadal cells or tissues in a subject in needthereof.
 11. A method of promoting, enhancing or improvingfertility-associated cell or tissue yield as part of an in vitrofertilization protocol, comprising contacting said fertility-associatedcell or tissue with a compound represented by the structure of formulaI:

wherein Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and R₁₀ is nothing, H, D, OH, halogen, oxo, nitro,CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone,—COOH, ester, trifluoromethyl, amide, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl,alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceuticallyacceptable salt, pharmaceutical product, hydrate, N-oxide, crystal orany combination thereof.
 12. Use of a compound represented by thestructure of formula I:

wherein Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido. alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and R₁₀ is nothing, H, D, OH, halogen, oxo, nitro,CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone,—COOH, ester, trifluoromethyl, amide, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl,alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceuticallyacceptable salt, pharmaceutical product, hydrate, N-oxide, crystal orany combination thereof for promoting, enhancing or improvingfertility-associated cell or tissue yield as part of an in vitrofertilization protocol.
 13. The method or use of any one of claims 1-12,wherein said compound is represented by the structure of formula IV:

wherein R₁, R₃, R₄, R₆, R₇, R₉ and R₁₀ are as described above.
 14. Themethod or use of any one of claims 1-13, wherein said compound isrepresented by the structure of formula VI:

wherein R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are the same or differentcomprising halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy,monoalkylamino, dialkylamino or arylamino; and R₁₀ is as describedabove.
 15. The method or use of any one of claims 1-14, wherein saidcompound is represented by the structure of formula VII:


16. The method or use of any one of claims 1-14, wherein said compoundis represented by the structure of formula VIII:


17. The method or use of any one of claims 1-14, wherein said compoundis represented by the structure of formula IX:


18. The method or use of any one of claims 1-14, wherein said compoundis represented by the structure of formula X:


19. The method or use of any one of claims 1-14, wherein said compoundis represented by the structure of formula XI:


20. The method or use of any one of claims 1-14, wherein said compoundis represented by the structure of formula XII:


21. The method or use of any one of claims 1-14, wherein said compoundis represented by the structure of formula XIII:


22. The method or use of any one of claims 1-12, wherein said compoundis represented by the structure of formula XIV:


23. The method or use of any one of claims 1-12, wherein said compoundis represented by the structure of formula XV:


24. The method or use of any one of claims 1-12, wherein said compoundis represented by the structure of formula XVI:


25. The method or use of any one of claims 1-612 wherein said compoundis represented by

or a combination thereof.
 26. The method or use of any one of claims1-25, wherein said cell or tissue is contacted with a pharmaceuticalcomposition comprising said compound.
 27. The method or use of any oneof claims 1-25, wherein said cell or tissue is isolated from or within asubject having a cancerous or precancerous condition, suffering from anendocrine disorder that negatively impacts steroidogenesis, sufferingfrom infertility or predisposed to infertility, or suffering from agenetic disorder or other disorder causing premature failure of agonadal cell or tissue.
 28. The method or use of any one of claims 1-25,wherein said cell or tissue is isolated from or within a subject havingbeen exposed to ionizing radiation.
 29. The method or use of any one ofclaims 1-26, wherein said subject is undergoing AIF, GIF or IVF.